Human mononuclear leukocytes have insulin receptors on their cell surface membrane. We have shown that the membrane receptors of the cultured human lymphoblastoid cell are independent of each other and that the negative cooperativity hormone binding model can no longer be considered a major regulatory mechanism of hormone binding. We have purified this radiolabeled detergent-solubilized receptor 300-fold to 10-15% purity and have found it to be a highly asymmetric glycoprotein of molecular weight 375,000. In the proposed continuation of this project, this receptor will be further purified to homogeneity by immunoabsorption methods, using anti-receptor antibodies from the serum of a patient with autoimmune insulin resistance. The radiolabeled receptor will be further characterized with respect to its binding stoichiometry (valence) by (1) affinity chromatography on agarose-insulin columns and (2) changes in molecular weight associated with the binding of dextran-insulin complexes (molecular weight 70,000) at high concentration. The thermodynamics of insulin binding to the solubilized receptor will be determined from the temperature dependence of the binding reaction, yielding the binding enthalpy and entropy as a function of temperature. The degree of hydrophobic interactions involved in the binding reaction will be estimated from the heat capacity change associated with binding and its temperature dependence. Insulin-receptor interactions will be studied by examining the changes in fluorescence polarization associated with the binding of fluorescein isothiocyanate-insulin to the detergent solubilized receptor. Changes in membrane microviscosity associated with the binding of insulin to the membrane receptor will be investigated by following changes in the fluorescence polarization of the fluorescent membrane probe diphenyl hexatriene. The above characterization of insulin binding to the human insulin receptor will elucidate the primary step in the mechanism of insulin action in humans.